Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants

Abstract

Streptococcus pyogenes Cas9 (SpCas9) variants with relaxed PAM requirements capable of high-resolution editing for various applications, and methods of use thereof.

What is claimed is: 
     
       1. An isolated  Streptococcus pyogenes  Cas9 (SpCas9) protein, comprising an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1, wherein the amino acids at positions: 1135, 1136, 1218, 1219, 1335, and 1337 are: LWKQQR (“SpG”); LWRQQR; LWSQQR; LWKHQR; LWKSQR; LWRSQR; LWRSQK; LWSHQR; LWRHQR; LWRQQK; LWSQQK; LSKQQR; LWKQQK; LSKHQR; LWKSQK; LSRHQR; LWRVQK; LFRQQR; LSRQQR; LSRSQR; LARQQR; LSRVQR; ASREQR; WSREQR; LSREQR; FSREQR; LSKSQR; LWKVQK; LWKHQK; LWSSQK; LWSHQK; LWSSQR; LWRVQR; LSKVQR; LWRHQK; LSSQQR; LWKVQR; LWSVQK; LSSHQR; LWSVQR; LSSVQR; LSKQQK; LSRVQK; LSKVQK; LSSSQR; LSKSQK; LSSVQK; LSRQQK; LSSQQK; LSRSQK; or LSKHQK, respectively. 
     
     
       2. The isolated SpCas9 protein of  claim 1 , further comprising a mutation at R1333, wherein R is mutated to P, C, A, V, G, K, L, S, T, Y, Q, I, H, N, M, D, E, F, or W. 
     
     
       3. The isolated SpCas9 protein of  claim 2 , wherein the amino acids at positions: 1135, 1136, 1218, 1219, 1333, 1335, and 1337 are: LWKQPQR; LWKQCQR; LWKQAQR; LWKQVQR; LWKQGQR; LWKQSQR; LWKQTQR; LWKQKQR; LWKQLQR; LWKQYQR; LWKQQQR; LWKQIQR; LWKQHQR; LWKQNQR; LWKQMQR; LWKQDQR; LWKQEQR; LWKQFQR; or LWKQWQR, respectively, in SEQ ID NO:1. 
     
     
       4. The isolated SpCas9 protein of  claim 1 , further comprising a mutation at N1317, wherein N is mutated to R, K, or H; G1104, wherein G is mutated to K, H, or R; A61, wherein A is mutated to R, K, H; L1111, wherein L is mutated to R or K, and/or A1322, wherein A is mutated to R or K. 
     
     
       5. The isolated SpCas9 protein of  claim 4 , comprising amino acids LWKQPQR at positions 1135, 1136, 1218, 1219, 1333, 1335, and 1337, respectively, in SEQ ID NO:1, and further comprising one of the following sets of mutations:
 A61R+N1317R+L1111R+A1322R (“SpRY”); 
 G1104K+N1317R+L1111R+A1322R; 
 A61R+G1104K+N1317R+L1111R+A1322R; 
 A61R+G1104K+L1111R+A1322R; 
 A61R+N1317R+L1111R+A1322R; 
 G1104K+L1111R+A1322R; 
 N1317R+L1111R+A1322R; or 
 A61R+L1111R+A1322R. 
 
     
     
       6. The isolated SpCas9 protein of  claim 1 , further comprising: (i) a mutation at a position selected from the group consisting of D10, E762, D839, H983, or D986; and/or
 (ii) a mutation at position H840 or N863. 
 
     
     
       7. The isolated SpCas9 protein of  claim 6 , wherein the mutations are:
 (i) D10A or D10N, and/or 
 (ii) H840A, H840N, or H840Y. 
 
     
     
       8. The isolated SpCas9 protein of  claim 1 , further comprising one or more mutations that increase specificity selected from the group consisting of mutations at N497, R661, N692, M694, Q695, H698, K810, K848, Q926, K1003, R0160, R691, M495, Y515, K526, R661, and combinations thereof. 
     
     
       9. The isolated SpCas9 protein of  claim 8 , further comprising mutations at R691A, M495V, Y515N, K526E, R661Q, R661L, R661S, Y450A/Q695A, L169A/Q695A, Q695A/Q926A, Q695A/D1135E, Q926A/D1135E, Y450A/D1135E, L169A/Y450A/Q695A, L169A/Q695A/Q926A, Y450A/Q695A/Q926A, R661A/Q695A/Q926A, N497A/Q695A/Q926A, Y450A/Q695A/D1135E, Y450A/Q926A/D1135E, Q695A/Q926A/D1135E, L169A/Y450A/Q695A/Q926A, L169A/R661A/Q695A/Q926A, Y450A/R661A/Q695A/Q926A, N497A/Q695A/Q926A/D1135E, R661A/Q695A/Q926A/D1135E, and Y450A/Q695A/Q926A/D1135E; N692A/M694A/Q695A/H698A, N692A/M694A/Q695A/H698A/Q926A; N692A/M694A/Q695A/Q926A; N692A/M694A/H698A/Q926A; N692A/Q695A/H698A/Q926A; M694A/Q695A/H698A/Q926A; N692A/Q695A/H698A; N692A/M694A/Q695A; N692A/H698A/Q926A; N692A/M694A/Q926A; N692A/M694A/H698A; M694A/Q695A/H698A; M694A/Q695A/Q926A; Q695A/H698A/Q926A; G582A/V583A/E584A/D585A/N588A/Q926A; G582A/V583A/E584A/D585A/N588A; T657A/G658A/W659A/R661A/Q926A; T657A/G658A/W659A/R661A; F491A/M495A/T496A/N497A/Q926A; F491A/M495A/T496A/N497A; K918A/V922A/R925A/Q926A; or 918A/V922A/R925A; K855A; K810A/K1003A/R1060A; K848A/K1003A/R1060A; M495V/Y515N/K526E/R661Q; M495V/Y515N/K526E/R661L; or M495V/Y515N/K526E/R661S. 
     
     
       10. A fusion protein comprising the isolated SpCas9 protein of  claim 1 , fused to a heterologous functional domain. 
     
     
       11. The fusion protein of  claim 10 , wherein the heterologous functional domain is a transcriptional activation domain. 
     
     
       12. The fusion protein of  claim 11 , wherein the transcriptional activation domain is from VP16, VP64, rTA, NF-κB p65, or the composite VPR (VP64-p65-rTA). 
     
     
       13. The fusion protein of  claim 10 , wherein the heterologous functional domain is a transcriptional silencer or transcriptional repression domain. 
     
     
       14. The fusion protein of  claim 13 , wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID). 
     
     
       15. The fusion protein of  claim 13 , wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1). 
     
     
       16. The fusion protein of  claim 10 , wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA. 
     
     
       17. The fusion protein of  claim 16 , wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a TET protein. 
     
     
       18. The fusion protein of  claim 17 , wherein the TET protein is TET1. 
     
     
       19. The fusion protein of  claim 10 , wherein the heterologous functional domain is an enzyme that modifies a histone subunit. 
     
     
       20. The fusion protein of  claim 19 , wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), or histone demethylase. 
     
     
       21. The fusion protein of  claim 10 , wherein the heterologous functional domain is a base editor or a prime editor. 
     
     
       22. The fusion protein of  claim 21 , wherein the base editor is a DNA or RNA deaminase; or wherein the prime editor comprises a reverse transcriptase (RT) domain. 
     
     
       23. The fusion protein of  claim 22 , wherein the base editor is a cytosine or adenine deaminase domain, or activation-induced cytidine deaminase. 
     
     
       24. The fusion protein of  claim 10 , wherein the heterologous functional domain is a biological tether. 
     
     
       25. The fusion protein of  claim 24 , wherein the biological tether is MS2, Csy4 or lambda N protein. 
     
     
       26. The fusion protein of  claim 10 , wherein the heterologous functional domain is FokI. 
     
     
       27. An isolated nucleic acid encoding the isolated SpCas9 protein of  claim 1 . 
     
     
       28. A vector comprising the isolated nucleic acid of  claim 27 . 
     
     
       29. The vector of  claim 28 , wherein the isolated nucleic acid of  claim 27  is operably linked to one or more regulatory domains for expressing the isolated  Streptococcus pyogenes  Cas9 (SpCas9) protein. 
     
     
       30. An isolated host cell, comprising the nucleic acid of  claim 27 . 
     
     
       31. The host cell of  claim 30 , wherein the host cell is a mammalian host cell. 
     
     
       32. A method of altering the genome of a cell, the method comprising expressing in the cell, or contacting the cell with, the isolated protein of  claim 1 , and a guide RNA having a region complementary to a selected portion of the genome of the cell. 
     
     
       33. The method of  claim 32 , wherein the isolated protein comprises one or more of a nuclear localization sequence, cell penetrating peptide sequence, and/or affinity tag. 
     
     
       34. The method of  claim 33 , wherein the cell is a stem cell. 
     
     
       35. The method of  claim 34 , wherein the cell is an embryonic stem cell, mesenchymal stem cell, or induced pluripotent stem cell; is in a living non-human animal; or is in a non-human embryo. 
     
     
       36. A method of altering a double stranded DNA (dsDNA) molecule, the method comprising contacting the dsDNA molecule with the isolated protein of  claim 1 , and a guide RNA having a region complementary to a selected portion of the dsDNA molecule. 
     
     
       37. The method of  claim 36 , wherein the dsDNA molecule is in vitro. 
     
     
       38. The method of  claim 36 , wherein the isolated SpCas9 protein and RNA are in a ribonucleoprotein complex.